Advanced cancers represent one of the major causes of human death. No effective methods of treatment have been suggested so far. Cancer immunotherapy aims at destroying tumor cells by immunological mechanisms. Immunotherapy compared to conventional methods of cancer therapy like surgery, radiation, and chemotherapy, is much less toxic, and no serious complications have been described so far. In addition, immunotherapy has the potency to work at different stages of disease. At initial stages it could be a good supplementary treatment to surgical removal of primary tumors, aiming to prevent development of disseminated disease. At advanced stages of disease it could be the only means of treatment, as conventional methods are often ineffective.
The main function of the immune system is to identify and destroy foreign substances (antigens) that invade the organism. The immune system is able to discriminate “self” from “non-self”, and under normal conditions only to develop an immune response against foreign or “non-self” antigens. Even though cancer cells originate from the organisms own cells they are treated as foreign by the natural immune system. However, this natural immune response is not strong enough in order to block the appearance and growth of the tumor. The task of immunotherapy is to increase the ability of the immune system to recognize tumor cells and to develop effective mechanisms of tumor elimination. The two main questions in any specific immunotherapy are which antigens to target and to find the optimal antigen presentation to the immune system.
Tumor associated antigens (TAA) recognized by cytotoxic T lymphocytes (CTL) is the most efficient component to be targeted among known effector mechanisms in anti-tumor immunity. There are two major types of TAA: unique antigens, present only in very few tumors and therefore not useful as general targets, and shared or common TAA, present in many tumors. Three major groups of shared antigens are currently considered as potential targets for immunotherapy and have all been shown to induce generation of cytotoxic T lymphocytes. The three groups are: Cancer/testis antigens (CT antigens), Tumor over-expressed antigens, and Lineage-specific differentiation antigens.
CT antigens are encoded by cancer or germ line specific genes, representing one of the largest groups of shared tumor-associated antigens. CT antigens were originally discovered in melanomas but have also been found in many other human malignancies. Among normal tissue they are only expressed in testis and in some cases in placenta. Normal cells expressing these antigens lack expression of MHC molecules and therefore these antigens are normally not accessible for recognition by T lymphocytes.
This makes CT antigens very attractive targets for specific cancer immunotherapy. Recent clinical trials have demonstrated tumor regression in a significant number of melanomas and bladder cancer patients by targeting of one specific CT antigen ((Nishiyama et al 2001, Clin. Cancer Res., v. 7, pp. 23-31; Thurner et al., 1999, J. Exp. Med., v. 190, pp. 1669-1678)). CT antigen recognition by T cells has only been reported for some CT antigens and the corresponding peptide epitopes determined. However, all CT antigens could be considered potential targets for immunotherapy. A correlation of the expression of MAGE-A antigens and tumor progression has been found in a number of malignancies ((Brasseur et al., 1995, Int. J. Cancer, v. 63, pp. 375-380; Eura et al., 1995, Int. J. Cancer, v. 64, pp. 304-308; Katano et al., 1997, J. Surg. Oncol., v. 64, pp. 195-201; Patard et al., 1995, Int. J. Cancer, v. 64, pp. 60-64)). Another group of CT antigens, the MAGE-B antigens, shows a significantly lower tumor-specific expression than the MAGE-A antigens. A third group, the MAGE-C antigens, displays an expression pattern that resembles the pattern of the MAGE-A antigens. No CTL response against MAGE-C antigens has been reported yet.
Several non-MAGE proteins with the characteristics of CT antigens have been described. One of them, NY-ESO-1 is one of the most immunogenic tumor antigens identified to date. Clinical trials with peptide immunization of melanoma patients demonstrated stabilization of disease and regression of some metastases in some patients ((Jäger et al., 2000, Proc. Natl. Acad. Sci. U.S.A, v. 97, pp. 12198-12203)).
In contrast to CT antigens, tumor over-expressed antigens lack strictly tumor specific expression, as their expression could be detected in low levels in some normal tissue types other than testis. Development of immunotherapy to some of these antigens could be beneficial for cancer patients, and presently such antigens like CEA, p53, HER-2/Neu, MUC-1 and alpha-fetoprotein are being intensively investigated as possible targets in clinical trials. The group of tumor over-expressed antigens has only recently been used as targets in clinical trials, and thus data on the efficiency of induction of therapeutic immune responses are absent.
The lineage specific antigens, melanocyte differentiation antigens and prostate-associated antigens, have so far only been described for two types of human cancers: melanomas and prostate cancer. This group of antigens is expressed both in normal differentiated tissue and in two types of human cancers. In normal differentiated tissue these antigens very rarely induce an immune response, however, these proteins become immunogenic in cancer cells, and in the case of melanomas, it is possible to detect T-killer cells reactive against melanocyte differentiation antigens. A prevailing number of clinical trails directed against melanomas or prostate cancer employ targeting of differentiation antigens.
Among the groups of tumor associated antigens the most promising data using TAA as targets for immunotherapy were obtained with some of the MAGE proteins. However, especially in case of melanomas, the therapeutic effect was unstable, and some metastases continued to grow. These metastases were usually negative for expression of the MAGE antigens used for immunization ((Thurner et al., 1999, J. Exp. Med., v. 190, pp. 1669-1678)).
One possibility for inducing a polyvalent immune response is to employ whole tumor cells or material derived from whole cells. WO 9003183, U.S. Pat. Nos. 5,840,317, and 6,187,306 describes several melanoma cell based vaccine preparations. U.S. Pat. No. 4,108,983 discloses a first generation melanoma vaccine derived from melanoma cells lysed by a vaccinia virus.
U.S. Pat. Nos. 5,635,188 and 5,030,621 disclose a vaccine of cell surface antigens from melanoma cells that are shed into the culture medium and subsequently used as an anti-melanoma vaccine. Similarly, U.S. Pat. No. 5,484,596 discloses a method of cancer therapy wherein irradiated tumor cells are injected into a human patient as a vaccine. U.S. Pat. No. 6,187,306 relates to melanoma cell lines expressing shared immunodominant melanoma antigens and methods of use.
An important aspect of any vaccine therapy is the way of vaccine administration. In recent years it has been realized that the most efficient way of antigen delivery to T cells, especially to naïve T cells, is by way of dendritic cells. Dendritic cells (DC) are the most efficient antigen presenting cells and DC based immunotherapy have already been used in different settings for treatment of cancer ((Kugler et al., 2000, Nat. Med., v. 6, pp. 332-336; Nestle et al., 1998, Nature (Med.), v. 4, pp. 328-332; Thurner et al., 1999, J. Exp. Med., v. 190, pp. 1669-1678)) demonstrating high potency of this way of immunization.
One of the unique properties of DC is their ability to uptake exogenous proteins by endocytosis, which are then processed and presented as peptide epitopes on their surface in conjunction with MHC class I antigens. The antigen presenting dendritic cells can be recognized by cytotoxic T cells. This property is extremely important when tumor cell antigens are applied in form of tumor lysates or apoptotic bodies added exogenously. High endocytic activity is believed to be associated with the immature state of DC differentiation based on comparison of immature and mature DC ((Sallusto et al., 1995, J. Exp. Med., v. 182, pp. 389-400)). The possibility that differences in the endocytic activity among immature DC could exist has never been considered.
WO 0127245 discloses a method of obtaining dendritic precursor cells from peripheral blood by standard leukapheresis, buoyant density gradient centrifugation and culture of the cells ex vivo in serum free medium for 40 hours in the absence of exogenously added cytokines.
WO 0146389 relates to a method for generating dendritic cells from leukapheresis products in closed systems, by using culture medium devoid of non-human proteins. Clinical grade cytokines (IL-4 and GM-CSF) are used in the culture medium and TAA are added for loading of the DC's.
Thurner et al., 1999, J. of Immunological Methods 223: 1-15, relates to a method for reproducible generation of large numbers of mature DC's from CD14+ monocytes by a two step method where cytokines are added after day 1.
The combination of dendritic cells with TAA's is also disclosed in WO 0128583, which relates to an immunotherapeutic vaccine providing antigen presenting cells mat have been pulsed with a disrupted cell preparation which includes cell membranes of cancer cells infected with recombinant vaccinia virus encoding at least one immunostimulating molecule. Also included is autologous DC's that presents a mixture of antigens from melanoma cell lines infected with a recombinant vaccinia virus encoding IL-2. In WO 0129192 a method is disclosed for inducing a tumor specific immune response in a patient, wherein antigen presenting cells from the patient are incubated with dead cell portions possessing at least one tumor antigen and the resulting loaded antigen presenting cells are administered to the patient.
Although the immunotherapeutic vaccines for treatment of cancer have improved over the recent years a need still exists for safer and more efficient compositions for use in cancer immunotherapy. Such vaccines should be polyvalent targeting several CT antigens to avoid outgrowth of antigen loss variants. They should be safer without the potential risk of targeting antigens expressed in normal tissue. They should be optimized for the presentation and delivery of the antigens to the T cells and also care should be taken to select the most efficient TAA's as targets.